The invention relates to methods for growing and quantifying the infectious or antigenic amount and determining antibodies against circoviruses, in particular porcine circoviruses (PCV). The invention also relates to methods for detecting the reduction in circoviruses and circovirus-like viruses of humans through the method for manufacturing pharmaceuticals from biological material (1).
Porcine circoviruses are isometric non-enveloped viruses about 17 nm in size and having single-stranded circular DNA of 1.76 kb. Antibodies against porcine circovirus have been found in serum from humans, mice and cattle by means of an indirect immunofluorescence detection (IFA) and by means of the ELISA method (2). Antibodies are detected in 53 to 92% of slaughter pigs (3). The clinical significance of PCV is still unknown. A new type or porcine circovirus, which leads to lesions in pig tissues and can be correlated with disease symptoms, has recently been detected. This so-called type 2 (PCV2) differs from the aforementioned type 1 (PCV1) mainly in terms of the pathogenicity (3 and 4).
Although porcine circovirus can be detected as contaminating agent in pig tissue cultures, it has not to date been possible to grow the virus in vitro and establish a routine test for growth and for quantitative detection of the virus, e.g. on the basis of a cytopathogenic effect (CPE), in the virus-replicating cells (2).
However, PCV is detected in vivo by means of the polymerase chain reaction in lymph nodes and cells of the spleen, tonsils, liver, heart, lungs, nasal mucosa, kidneys, pancreas and intestine (3). Since pig organs are used for human transplantation (5), porcine circovirus should be regarded as a potential risk virus for humans.
The object of the present invention was therefore to develop a method for cultivating porcine circovirus in vitro in order to be able to examine its infectivity. It was additionally intended to develop a method for neutralizing porcine circovirus by specific antibodies and removing it from biological materials from pigs, humans or other vertebrates, so that these materials can be employed without reservations directly or indirectly for therapeutic purposes, e.g. for obtaining insulin, heparin, blood and plasma, cell culture media and constituents thereof, including trypsin, and for cells for producing recombinant proteins. It was finally intended that successful growing of PCV in cell cultures would also make it possible to produce a vaccine by methods known per se. This may involve using inactivated PCT or an avirulent PCV strain (e.g. through selection of an avirulent PCV strain after adaptation to various cell cultures and/or after treatment of infected cell cultures with mutagens or after genetic modification of the PCV) as live vaccine. In addition, the antigenic material obtained from grown porcine circoviruses can also be employed for diagnostic purposes.
This object is achieved by a method for growing circoviruses, in particular porcine circoviruses (PCV), in which circoviruses obtained from an infected cell culture grow in cultures of porcine, bovine or human cells after one or more passages in the cell cultures.
For the method of the invention, porcine circoviruses obtained from a PK15 (=porcine kidney) cell culture inapparently infected with PCV were, after passage in other cell cultures, in particular porcine cell cultures, grown in these cell cultures, with a cytopathogenic effect being displayed. The presence of porcine circoviruses was in this case verified with the aid of a specific nested polymerase chain reaction (PCR). Primers with the following DNA sequences were employed for this:
1. PCR plus: GAG AGG AAG GTT TGG AAG AGG (946-966) (SEQ ID NO. 1)
1. PCR minus: CCA CTG GCT CTT CCC ACA ACC (1358-1338) (SEQ ID NO. 2). 
2. PCR plus: GGT GAA GTG GTA TTT TGG TGC C (1025-1046) (SEQ ID NO. 3)
2. PCR minus: CTA TGA CGT GTA CAG CTG TCT TCC (1326-1303) (SEQ ID NO. 4)
These primers were selected from the origin of replication (7).
When carrying out the method of the invention, it was observed that the porcine circoviruses cannot be grown equally well in all cultures of various mammalian and human cells. Growth was successful in cultures of cells from various porcine organs, from bovine kidney, bovine lung and human lung. A cell culture which is very suitable for successful growth of porcine circovirus and which was developed from fetal porcine testis was deposited on Jul. 10, 2000, under the provisions of the Budapest Treaty at Deutsche Sammiung Von Mikroorganismen und Zelikulturen GmbH (DSMZ), Mascheroder Weg 1b, Dxe2x80x9438124 Braunschweig, German, and was assigned Accession No. FSHO-DSM ACC 2466.
The ELISA method is very suitable for quantitative detection of antibodies present in serum against porcine circovirus. This entails circoviruses being incubated, after adsorption onto a support material, with the serum to be investigated and thus being bound to a primary antibody present in the serum. A secondary labeled antibody directed against the primary antibody is then brought into contact therewith and, after the unbound secondary antibody has been washed out, the light signal emitted by the bound labeled antibody (extinction) is measured. The sandwich method known to the skilled worker is suitable for detecting the circovirus antigen, in which case an antibody against circoviruses which is bound to a support material binds the antigen in the serum to be investigated; a (labeled) antibody directed against circovirus antigen is then brought into contact therewith and, after the unbound antibody has been washed out, the light signal emitted by the bound labeled antibody (extinction) is measured.
Sera containing neutralizing antibodies against porcine circovirus are suitable for neutralizing circoviruses in biological material. Neutralizing antibodies have been found in porcine sera and human immunoglobulin (xcex3-globulin). A human immunoglobulin particularly suitable for neutralizing circoviruses in biological material is one obtained from high-titer human donors who have a titer of specific antibodies which is at least two to three times higher than normal donors, the average PCV antibody titer being determined on a plasma pool from at least 1000 donors.
The virus safety of pharmaceuticals of biological origin (e.g. from human blood/plasma, from cell cultures, from animal organs/tissues) is investigated as required by the authorities (e.g. CPMP/BWP/268/95: Note for guidance on virus validation studies: the design, contribution and interpretation of studies validating the inactivation and removal of viruses; CPMP/BWP/269/95 rev. 3: Note for guidance on plasma-derived medicinal products); in these so-called virus validation studies, viruses are deliberately added to material at various production steps in the method of manufacture of biologicals, and the removal and/or inactivation of the viruses by the step in the method is determined. The viruses used for this investigation either should possibly occur in the biological starting material or, if these viruses cannot be grown in vitro, are model viruses with physico-chemical properties as similar as possible to the contaminating viruses. An example of a model virus for the human circovirus TTV is PCV. In investigations of a step in the method of manufacture of therapeutic compositions from biological materialxe2x80x94heat treatment at 60xc2x0 C. in stabilized aqueous solutionxe2x80x94it emerged that the porcine circovirus is labile and could be inactivated within a few hours; it is thus possible to demonstrate the capacity of the method of manufacturing biologicals to inactivate TTV by a heat treatment. It is possible analogously to investigate other steps in the manufacturing process for the ability to remove TTV (e.g. by precipitation, adsorption or chromatography or filtration steps) or inactivate TTV (e.g. by chaotropic salts or substances which intercalate in nucleic acids, or by irradiation with high-energy rays) using PCV. It is additionally possible to establish the capacity for inactivating and/or removing viruses also, for example, for additives in the production of pharmaceuticals, such as, for example, sera and other ingredients of media for cell cultures for producing recombinant proteins or monoclonal antibodies for affinity chromatography for purifying and concentrating active ingredients.